Development and validation of a label-free method for measuring the collagen hydrolytic activity of protease.

Collagen is the most abundant fibrous structural protein, and therefore, the quantitative evaluation of the effect of protease on collagen has a profound influence on enzyme application. In this research, unlabeled native bovine hide powder was utilized to detect collagen hydrolytic activity of the protease. The optimum conditions of the determination method were as follows: 30 mg/mL substrate concentration, 30 min reaction time, and 2-9 U/mL enzyme concentration. Then, several typical industrial protease preparations were chosen to measure collagenolytic activities at different temperatures and pH values, whose change trends were quite distinct from those of proteolytic activity assay method based on casein or dye-labeled hide powder substrate. Especially, in the pH 5-7, casein hydrolytic activities of these proteases showed sharper peaks with relative activity from 6% to 100%, whereas, their collagen hydrolytic activities based on native hide powder exhibited 30-100% with broader peaks. And collagen hydrolytic activities resulted from using dye-labeled substrate reached a lower optimum pH value than that of other methods. Besides, the results of these measurements displayed a moderate degree of reproducibility. This method is more reasonable than the protease assay method using casein or labeled hide powder as the substrate in many fields.